Human immunodeficiency virus (HIV-1) enters target cells by means of sequential binding of its viral exterior envelope glycoprotein, gp120, to CD4 and a chemokine receptor, CCR5 or CXCR4. Binding of gp120 to CD4 exposes so-called CD4-inducible (CD4i) epitopes on gp120. These epitopes include a highly conserved beta-strand important for binding to the chemokine receptors. In vitro, the level of coreceptor expression by the target cell and the size of neutralizing antibodies targeting CD4i epitopes influence the neutralizing potency. These observations suggest that exposure of the relevant CD4i epitopes may be sterically restricted in primary isolates and has prompted speculations that these epitopes may have limited utility as vaccine and therapeutic targets. Although numerous neutralizing CD4i antibodies have been identified, the relevance of these epitopes in mitigating infection has never been established in a formal passive protection challenge study. We propose to perform such a study using the CD4i antibody, m9, in conjunction with a rectal challenge with SHIV162P3. M9 is a human broadly neutralizing antibody that recognizes a highly conserved CD4i epitope and is most effective as a scFv antibody fragment. We have engineered a scFv-m9 variant with significantly improved pharmacokinetic profile in Cynomolgus macaque as well as an IgG1 version of m9. Comparing the protective efficacy afforded by the modified scFv-m9 and the IgG1-m9 in the proposed challenge study will confirm or reject the hypothesis that the steric interactions, i.e., the size, of antibodies targeting the CD4i epitopes impacts their efficacy in vivo as it does in vitro. The proposed study will explore the vaccine utilities of the CD4i epitopes via the following 3 Aims: (1) Engineer variants of scFv-m9 that have Fc-mediated antiviral activity;(2) Produce the necessary quantities of selected scFv-m9 variant and IgG1-m9 for challenge studies in Aim 3;(3) Assess the efficacy of passively administered scFv-m9 variant vs IgG1-m9 against a rectal challenge with SHIV162P3 in rhesus macaques. Protection in the group receiving IgG1-m9 will indicate that CD4i antibodies are effective in vivo independently of their poor neutralization efficacy in vitro, which will enable vaccine studies targeting CD4i epitopes. Comparisons of efficacy provided by the IgG1 and scFv versions of m9 will evaluate the anti-HIV role of the steric restrictions around CD4i epitopes in vivo. Such information will inform and enable development of effective vaccines against HIV.